ERYTHROCYTES AS DRUG DELIVERY SYSTEM:

A BOON TO CURE
Toshika Jain*, Rohit Adhav, Priyanka Vaswani
Shri G. S. Institute Of Technology and Sciences Indore
Madhya Pradesh, India.
Abstract
The biocompatibility, non-immunogenicity, non-pathogenicity and biodegradability make the
erythrocytes as unique and useful carriers. Erythrocytes are non-toxic and biocompatible with human
body. The specific and accurate amount of drug will be incorporated into the cells by causing osmotic
manipulation in cell structure. Biopharmaceuticals, therapeutically significant peptides and proteins,
nucleic acid-based biological, antigens, anticancer drug and Vaccines are among the recently focused
pharmaceuticals for being delivered using carrier Erythrocytes. So many drugs like aspirin, steroid,
cancer drug which have many side effects are reduced by use of resealed Erythrocytes. Current
review highlights isolation, drug loading methods, evaluation methods and applications of resealed
Erythrocytes for drug delivery.
Keywords: Erythrocytes, Drug Delivery System, Manipulation, Biopharmaceutical.
1. Introduction
Blood contains different type of cells like Erythrocytes (RBC), Leucocytes (WBC)
and Platelets. Among them, Erythrocytes are the most interesting carrier and possess great potential in
drug delivery due to their ability to circulate throughout the body, zero order kinetics, reproducibility
and ease of preparation. Primary aim for the development of this drug delivery system is to maximize
therapeutic performance, reducing undesirable side effects of drug as well as increase patient
compliance [9]. The overall process is based on the response of these cells under osmotic conditions.
Upon reinjection, the drug-loaded erythrocytes serve as slow circulating depots and target the drugs to
disease tissue or organ. Present pharmaceutical scenario is aimed at development of drug delivery
systems which maximize the drug targeting along with high therapeutic benefits for safe and effective
management of diseases. Targeting of an active bio molecule from effective drug delivery where
pharmacological agent directed specifically to its target site. Drug targeting can be done by
approaches as either chemical modification or by appropriate carrier.
2. Erythrocytes
Red blood cells (also referred to as erythrocytes) are the most common type of blood
cells and the vertebrate organism's principal means of delivering oxygen (O 2) to the body tissues via
the blood flow through the circulatory system. The cells develop in the bone marrow and circulate for
about 100120 days in the body before their components are recycled by macrophages. Each
circulation takes about 20 seconds. Approximately, quarters of the cells in the human body are red
blood cells.
3. Physiology
Red blood cells are highly specialized for their oxygen transport function. Because
mature RBCs have no nucleus, all their internal space is available for oxygen transport. Because
RBCs lack mitochondria and generate ATP anaerobically (without oxygen), they do not use up any of
the oxygen they transport. Even the shape of RBC facilitates its function. A biconcave disc has a

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much greater surface area for the diffusion of gas molecules in to and out of the RBC than would, say,
a sphere or a cube. Each RBC contains about 280 million hemoglobin molecules.
A hemoglobin molecule consists of a protein called globin, composed of four
polypeptide chains (two alpha and two beta chains); a ring like non protein pigment called a heme
(Figure 1) is bound to each of the four chains. At the center of eachheme ring is an iron ion that can
combine reversibly with one oxygen molecule (Figure 1), allowing each hemoglobin molecule to bind
four oxygen molecules. Each oxygen molecule picked up from the lungs is bound to an iron ion. As
blood flows through tissue capillaries, the ironoxygen reaction reverses. Hemoglobin releases
oxygen, which diffuses first into the interstitial fluid and then into cells.
Hemoglobin also transports about 23% of the total carbon dioxide, a waste product of
metabolism. Blood flowing through tissue capillaries picks up carbon dioxide, some of which
combines with amino acids in the globin part of hemoglobin. As blood flows through the lungs, the
carbon dioxide is released from hemoglobin and then exhaled. In addition to its key role in
transporting oxygen and carbon dioxide, hemoglobin also plays a role in the regulation of blood flow
and blood pressure.
The gaseous hormone Nitric oxide (NO), produced by the endothelial cells that line
blood vessels, binds to hemoglobin. Under some circumstances, hemoglobin releases NO. The
released NO causes vasodilatation, an increase in blood vessel diameter that occurs when the smooth
muscle in the vessel wall relaxes. Vasodilation improves blood flow and enhances oxygen delivery to
cells near the site of NO release.
4. Source of Erythrocytes
Various types of mammalian Erythrocytes have been used for drug delivery, including
erythrocytes of mice, cattle, pig, dog, sheep, goat, monkey, chicken, rat, and rabbit.
5. Isolation of Erythrocytes
Erythrocytes may be prepared as carriers from blood taken from human beings and
from different animal species, such as rat, mice, rabbit, dog, etc. [8]. Blood is taken from the human
being, mouse, rat or the animal species in question, using a suitable anti-coagulant. Application is
normally made of EDTA, as it is the anticoagulant that best preserves the properties of blood cells.
Freshly collected blood is centrifuged in a refrigerated centrifuge in order to separate packed
Erythrocytes. Several washes are subsequently performed. This is a process that normally involves
repeated centrifugation with an isosmotic solution to remove other blood components. The
Erythrocytes can be washed more efficiently by using a capillary hollow fiber plasma separator. The
hematocrits employed may be variables ranging between 5 % and 95 %, although the most usual is to
work with a hematocrit of 70 %. In 1953, Gardos tried to load Erythrocyte ghost using Adenosine
triphosphate (ATP). In 1959, Marsden and Ostting, reported the entrapment of dextran (molecular
weight 10250 kDa). In 1973, the loading of drugs in Erythrocytes was reported separately by Ihler et
al. and Zimmermann. In 1979, the term carrier Erythrocytes was coined to describe drug-loaded
Erythrocytes [1], [3], [7].
6. Methods of Drug Loading
Several methods have been reported for encapsulation of drug or other bioactive
agents in Erythrocytes. Some of these methods such as electrical pulse methods and osmosis-based
methods have a physical nature whereas the other methods such as the chemical perturbation of the

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Figure 1

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membrane are chemically based. Regardless of the method used, the optimal characteristics for a
compound to be encapsulated successfully in Erythrocytes include a considerable degree of water
solubility, resistance against inactivation within the Erythrocytes, the lack of physical and/or chemical
interaction with Erythrocyte membrane or the other cell constituents, and well-defined
pharmacokinetic as well as pharmaco-dynamics properties. Hypotonic hemolysis, hypotonic dilution,
hypotonic dialysis, hypotonic pre swelling, and osmotic pulse methods are categorized as osmosisbased methods. Chemical perturbation of the membrane, electrical breakdown or electroporation,
endocytosis, lipid fusion, laser loading, and intrinsic uptake of substances by Erythrocytes are other
reported methods used for encapsulation of drugs and other agents into Erythrocytes.
7. Hypotonic Hemolysis
This method is based on the ability of Erythrocytes to undergo reversible swelling in
a hypotonic solution. Erythrocytes have an exceptional capability for reversible shape changes with or
without accompanying volume change and for reversible deformation under stress. An increase in
volume leads to an initial change in the shape from biconcave to spherical. This change is attributable
to the absence of superfluous membrane; hence the surface area of the cell is fixed. The cells assume a
spherical shape to accommodate additional volume while keeping the surface area constant. The
volume gain is 2550 %. The cells can maintain their integrity up to a tonicity of ~150 moss/kg,
above which the membrane ruptures, releasing the cellular contents. At this point (just before cell
lysis), some transient pores of 200500 are generated on the membrane. After cell lysis, cellular
contents are depleted. The remnant is called an Erythrocyte ghost. The principle of using these
ruptured Erythrocytes as drug carriers is based on the fact that the ruptured membranes can be
resealed by restoring isotonic conditions. Upon incubation, the cells resume their original biconcave
shape and recover original impermeability.
8. Use of Red Cell Loader
A novel method for entrapment of non-diffusible drugs into Erythrocytes is by red
cell loader method. With as little as 50 ml of a blood sample, different biologically active compounds
were entrapped into Erythrocytes within a period of 2 hours at room temperature under blood banking
conditions. The process is based on two sequential hypotonic dilutions of washed Erythrocytes
followed by concentration with a hem filter and an isotonic resealing of the cells. There was ~30 %
drug loading with 35-50 % cell recovery. The processed Erythrocytes had normal survival in vivo.
The same cells could be used for targeting by improving their recognition by tissue macrophages.
9. Hypotonic Dilution
Hypotonic dilution was the first method investigated for the encapsulation of
chemicals into Erythrocytes and is the simplest and fastest method. In this method, a volume of
packed Erythrocytes is diluted with 220 volumes of aqueous solution of a drug. The solution tonicity
is then restored by adding a hypertonic buffer. The resultant mixture is then centrifuged, the
supernatant is discarded, and the pellet is washed with isotonic buffer solution. The major drawbacks
of this method include low entrapment efficiency and a considerable loss of hemoglobin and other cell
components. This reduces the circulation half-life of the loaded cells. These cells are readily
phagocytosis by RES (Reticulo-endothelial system) macrophages and hence can be used for targeting
RES organs. Hypotonic dilution is used for loading enzymes such as -galactosidase and glucosidase, asparginase and arginase, as well as bronchodilators such as salbutamol.
10. Hypotonic Pre-swelling
This method was developed by Rechsteinerin in 1975 and was modified by Jenner et
al. for drug loading. The technique is based upon initial controlled swelling in a hypotonic buffered
solution. This mixture is centrifuged at low g values. The supernatant is discarded and the cell fraction
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is brought to the lysis point by adding 100120 l portions of an aqueous solution of the drug to be
encapsulated. The mixture is centrifuged between the drug-addition steps [10]. The lysis point is
detected by the disappearance of a distinct boundary between the cell fraction and the supernatant
upon centrifugation. The tonicity of a cell mixture is restored at the lysis point by adding a calculated
amount of hypertonic buffer. Then, the cell suspension is incubated at 37 C to reanneal the resealed
Erythrocytes. Such cells have a circulation half-life comparable to that of normal cells [6], [11]. This
method is simpler and faster than other methods, causing minimum damage to cells. Drugs
encapsulated in Erythrocytes using this method include propranolol, asparginase, cyclopohphamide,
cortisol-21-phosphate, 1-antitrypsin, metronidazole, levothyroxine, methotrexate, insulin, enalaprilat
and isoniazid [8].
11. Hypotonic dialysis
This method was first reported by Klibanskyin 1959 and was used in 1977 by
Deloach and Ihler, and Dale for loading enzymes and lipids [2]. Several methods are based on the
principle that semi permeable dialysis membrane maximizes the intracellular: extracellular volume
ratio for macromolecules during lysis and resealing. In the process, an isotonic, buffered suspension
of Erythrocytes with a hematocrit value of 7080 is prepared and placed in a conventional dialysis
tube immersed in 1020 volumes of a hypotonic buffer [4], [10]. The medium is agitated slowly for 2
hours [4]. The tonicity of the dialysis tube is restored by directly adding a calculated amount of a
hypertonic buffer to the surrounding medium or by replacing the surrounding medium by isotonic
buffer [7].The drug to be loaded can be added by either dissolving the drug in isotonic cell suspending
buffer inside a dialysis bag at the beginning of the experiment or by adding the drug to a dialysis bag
after the stirring is complete. The use of standard hemodialysis equipment for loading a drug in
Erythrocytes was reported by Roper et al. In this method, the Erythrocyte suspension and the drug to
be loaded were placed in the blood compartment and the hypotonic buffer was placed in a receptor
compartment [5], [6], and [9]. This led to the concept of continuous flow dialysis, which has been
used by several other researchers. The loaded cells exhibit the same circulation half life as that of
normal cells. Also, this method has high entrapment efficiency on the order of 30-50 %, cell recovery
of 70-80 %, high-loading capacity, and is amenable to automation with control of process variables.
The drawbacks include a long processing time and the need for special equipment [7], [10]. This
method has been used for loading enzymes such as -galactosidase, glucoserebrosidase , asparginase,
inositol hexaphosphatase, as well as drugs such as gentamicin, Adriamycin,pentamidine and
furamycin, interlukin-2, desferroxamine , and human recombinant erythropoietin [2], [ 5], [11].
12. Isotonic Osmoticlysis
This method, also known as the osmotic pulse method, involves isotonic hemolysis
that is achieved by physical or chemical means [2], [4]. The isotonic solutions may or may not be
isoionic. If Erythrocytes are incubated in solutions of a substance with high membrane permeability,
the solute will diffuse into the cells because of the concentration gradient. This process is followed by
an influx of water to maintain osmotic equilibrium. Chemicals such as urea solution, polyethylene
glycol, and ammonium chloride have been used for isotonic hemolysis [1], [3], [7]. However, this
method also is not immune to changes in membrane structure composition. In 1987, Franco et al.
developed a method that involved suspending Erythrocytes in an isotonic solution of Dimethyl
sulfoxide (DMSO).The suspension was diluted with an isotonic-buffered drug solution. After the cells
were separated, they were resealed at 37C [10].
13. Chemical perturbation of the Membrane
This method is based on the increase in membrane permeability of Erythrocytes when
the cells are exposed to certain chemicals. In 1973, Deuticke et al. showed that the permeability of
Erythrocytic membrane increases upon exposure to polyene antibiotic such as amphotericin B. In
1980, this method was used successfully by Kitao and Hattori to entrap the antineoplastic drug
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daunomycin in human and mouse Erythrocytes. Lin et al. used halothane for the same purpose.
However, these methods induce irreversible destructive changes in the cell membrane and hence are
not very popular.
14. Electro-insertion or Electro Encapsulation
In 1973, Zimmermann tried an electrical pulse method to encapsulate bioactive
molecules. Also known as electroporation, the method is based on the observation that electrical
shock brings about irreversible changes in an Erythrocyte membrane. In 1977, Tsong and Kinosita
suggested the use of transient electrolysis to generate desirable membrane permeability for drug
loading. The Erythrocyte membrane is opened by a dielectric breakdown. Subsequently, the pores can
be resealed by incubation at 37C in an isotonic medium. The procedure involves suspending
Erythrocytes in an isotonic buffer in an electrical discharge chamber, a capacitor and the
sophistication of the process. Entrapment efficiency of this method is ~35 %, and the life span of the
resealed cells in circulation is comparable with that of normal cells. Various compounds such as
sucrose, urease, methotrexate, isoniazid, human glycophorin, DNA fragments, and latex particles of
diameter 0.2 m can be entrapped within Erythrocytes by this method. Mangal and Kaur achieved
sustained release of a drug entrapped in Erythrocytes with the use of electroporation.
15. Entrapment by Endocytosis
This method was reported by Schrier et al. in 1975. Endocytosis involves the addition
of one volume of washed packed Erythrocytes to nine volumes of buffer containing 2.5 mm ATP, 2.5
mm MgCl2, and 1mm CaCl2, followed by incubation for 2 min. at room temperature. The pores
created by this method are resealed by using 154 mm of Na Cl and incubation at 37C for 2 min.
16. Loading by Electric Cell Fusion
This method involves the initial loading of drug molecules into Erythrocyte ghosts
followed by adhesion of these cells to target cells. The fusion is accentuated by the application of an
electric pulse, which causes the release of an entrapped molecule. An example of this method is
loading a cell-specific monoclonal antibody into an Erythrocyte ghost. An antibody against a specific
surface protein of target cells can be chemically cross-linked to drug-loaded cells that would direct
these cells to desired cells.
17. Loading by lipid fusion
Lipid vesicles containing a drug can be directly fused to human Erythrocytes, which
lead to an exchange with a lipid-entrapped drug. This technique was used for entrapping inositol
monophosphate to improve the oxygen carrying capacity of cells. However, the entrapment efficiency
of this method is very low (~1 %).
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[3]. Eichler, H.C., Gasic, S., Daum, B., Bacher, S. and Sreger, G. 1987; In vitro drug release from
human carrier erythrocytes, Adv. Biosci. 67: 1115.

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